Methods for Genotyping Selected Polymorphism

ABSTRACT

Methods for genotyping polymorphisms using a locus specific primer that is complementary to a region near a selected polymorphism are described. Methods for synthesizing pools of locus specific primers that incorporate some degenerate positions are also disclosed. A plurality of different sequence capture probes are synthesized simultaneously using degenerate oligonucleotide synthesis. The sequence of the locus specific regions of the capture probes are related in that they have some bases that are identical in each sequence in the plurality of sequences and positions that vary from one locus specific region to another. The sequences are selected based on proximity to a polymorphism of interest and because they conform to a similar sequence pattern.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. applicationSer. No. 10/264,945 filed Oct. 4, 2002 and claims priority to U.S.Provisional application No. 60/493,085, filed Aug. 5, 2003, the entiredisclosures of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to enrichment and amplification of a collection oftarget sequences from a nucleic acid sample and methods of analyzingamplified product. In some embodiments target sequences are amplified byextension of a locus-specific primer followed by amplification of theextended locus-specific primer with a generic pair of primers. In someembodiments the locus-specific primers are attached to a solid supportand extension takes place on the solid support. In some embodiments theinvention relates to the preparation of target for array based analysisof genotype. The present invention relates to the fields of molecularbiology and genetics.

BACKGROUND OF THE INVENTION

The past years have seen a dynamic change in the ability of science tocomprehend vast amounts of data. Pioneering technologies such as nucleicacid arrays allow scientists to delve into the world of genetics in fargreater detail than ever before. Exploration of genomic DNA has longbeen a dream of the scientific community. Held within the complexstructures of genomic DNA lies the potential to identify, diagnose, ortreat diseases like cancer, Alzheimer disease or alcoholism.Exploitation of genomic information from plants and animals may alsoprovide answers to the world's food distribution problems.

Recent efforts in the scientific community, such as the publication ofthe draft sequence of the human genome in February 2001, have changedthe dream of genome exploration into a reality. Genome-wide assays,however, must contend with the complexity of genomes; the human genomefor example is estimated to have a complexity of 3×10⁹ base pairs. Novelmethods of sample preparation and sample analysis that reduce complexitymay provide for the fast and cost effective exploration of complexsamples of nucleic acids, particularly genomic DNA.

SUMMARY OF THE INVENTION

A method of genotyping a plurality of polymorphisms present in targetsequences is disclosed. A pool of potential target polymorphisms isselected and the sequences near the polymorphism are analyzed toidentify a sequence for targeting a locus specific primer. The sequencecomprises a common sequence and a consensus sequence. The commonsequence is a stretch of at least 4 bases that are identical in eachtarget sequence. The consensus sequence comprises a region that has somebases that are identical in all target sequences and some bases that arevariable between target sequences. The polymorphisms to be analyzed maybe selected based on the presence of the common and consensus sequences.Preferably the common and consensus sequences are immediately adjacentto one another and more preferably the common sequence is between theconsensus sequence and the polymorphic position so that a primercontaining the consensus sequence 5′ of the common sequence can beextended in the direction of the polymorphic position. The consensussequence may be first identified by identifying a common sequence thatis present in many of the targets, such as a restriction enzymerecognition site and then analyzing the sequence that is immediatelyadjacent to the common sequence in a plurality of potential targetsequences. The sequences surrounding the common sequence are thenanalyzed to identify a target sequences that have similar sequenceimmediately adjacent to the common sequence. In this way a plurality oftargets, containing polymorphisms, are identified that can be hybridizedto a pool of primers or capture probes that are synthesized in the samedegenerate synthesis reaction or in a limited number of synthesisreactions that include pooled and separate monomer addition steps.

The pool of capture probes is hybridized to genomic DNA, which may beadaptor ligated fragment, and extended through the polymorphism. Theextended capture probes may be amplified, by PCR for example. Theamplification product may be hybridized to an array of probes designedto discriminate between different alleles of a polymorphic allele sothat the base or bases present at the polymorphic positions may bedetermined. An array comprising allele specific probes for thepolymorphic positions to be genotyped is also disclosed. The probes ofthe array are selected based on the target sequences that are selected.Hybridization to the array is analyzed to determine the bases present atthe polymorphic positions.

Kits for performing the disclosed methods are also disclosed. The kitsmay comprise pools of capture probes designed for amplification of aplurality of target sequences. The target sequences are selected so thatthey each contain a polymorphic position of interest, they each share acommon sequence and a consensus sequence that is immediately adjacent tothe common sequence, and the common sequence is within 1000 base pairsof the polymorphic position. The common sequence is the same in aplurality of targets and the consensus sequence has positions that areidentical in each target sequence and positions that are variablebetween target sequences. The capture probes are complementary to thecommon and consensus sequences and also comprises a universal primingsequence that is 5′ of the region that is complementary to the target.The capture probes may be pooled into containers that contain 2 or moredifferent sequence capture probes. Preferably 100 or more differentsequence capture probes are pooled into a single container. The captureprobes may be synthesized by combinatorial methods and may include stepswhere a mixture of bases is added. The kit may further compriseadaptors, universal primers, dNTPs, ligase, buffer, and polymerase.

The kits may be used to amplify a collection of target sequences.Amplification may be by fragmentation of the sample, ligation of anadaptor to the fragments, hybridization of capture probes to theadaptor-ligated fragments, extension of the capture probe, andamplification of the extended capture probes using a pair of universalprimers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a method of amplifying specific target sequences using acapture probe that is locus specific and genomic DNA that has beenligated to an adaptor. The capture probes are attached to a solidsupport and extended to incorporate the sequence of interest and theadaptor sequence. The extended capture probes are released from thesolid support and amplified with a single primer pair.

FIG. 2 shows a method where the capture probes are attached to a solidsupport by hybridization to a probe that is covalently attached to thesolid support. The probes on the array are complementary to a tagsequence in the 5′ region of the capture probe. The capture probehybridizes so that the 3′ end is available for extension.

FIG. 3 shows a schematic of solution-based multiplexed SNP genotyping. Asample is fragmented and ligated to an adaptor so that the adaptorsequence that hybridizes to the 3′ end of the strands of the fragmentsis blocked from extension. Locus specific capture probes are hybridizedto the fragments and extended in solution then amplified by PCR usingprimers to A1 and A2. Prior to amplification the extended capture probesmay be enriched by, for example, removal of non-extended products or bypositive selection of extended products.

FIG. 4 shows amplification of multiple loci with a degenerate captureprobe. A capture probe with degenerate positions (indicated by N) andconstant positions (indicated by G, A and T) is shown hybridizing tothree different loci, (locus 1, 2 and 3). The degenerate capture probealso has a 5′ common region which is the T7 promoter primer sequence.The three loci have a polymorphism (*) and a Not1 restriction site. Thecapture probes are hybridized to the target sequences and extended. Theextended products are digested with Not1 and adaptors are ligated to theends. The fragments are then amplified with primers to the adaptor andto the T7 sequence.

FIGS. 5A and 5B show schematics of combinatorial synthesis of degenerateprimers on an array. FIG. 5A shows an array of probes where the majorityof the probes in a feature have the same sequence and a plurality of thefeatures is designed have a common core sequence. Each feature of thearray has the sequence GNTNNAG and the N's are the same within a featureand different between features. FIG. 5B shows an array of probes wherethe probes in a feature have the same core sequence but are different atsome positions and the different features have different core sequences.Feature 1 has the core sequence GTGNTNNAG, feature 2 has the coresequence GTNNGNGTC and feature 3 has the core sequence ANGNNTACA.

FIGS. 6A and 6B show a comparison of synthesis methods. FIG. 6A showscombinatorial methods that include separate and pooled monomer additionsteps. FIG. 6B shows synthesis without the use of pooled monomeraddition steps.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS (A.) General

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

An individual is not limited to a human being but may also be otherorganisms including but not limited to mammals, plants, bacteria, orcells derived from any of the above.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. Ser. No.09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269,6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730(International Publication Number WO 99/36760) and PCT/US01/04285, whichare all incorporated herein by reference in their entirety for allpurposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptidearrays.

Nucleic acid arrays that are useful in the present invention includethose that are commercially available from Affymetrix (Santa Clara,Calif.) under the brand name GeneChip®. Example arrays are shown on thewebsite at affymetrix.com.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. Nos.60/319,253, 10/013,598, and U.S. Pat. Nos. 5,856,092, 6,300,063,5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses areembodied in U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061,and 6,197,506.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675, and each of which is incorporated herein by reference intheir entireties for all purposes. The sample may be amplified on thearray. See, for example, U.S. Pat. No. 6,300,070 and U.S. patentapplication Ser. No. 09/513,300, which are incorporated herein byreference.

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al.,Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), selective amplification of targetpolynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequenceprimed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975),arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos.5,413,909, 5,861,245), degenerate oligonucleotide primed PCR (DOP-PCR),(Telenius H., et al. Genomics 13:718-25 (1992)), self-sustained sequencereplication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)and WO90/06995) and nucleic acid based sequence amplification (NABSA).(See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, each of whichis incorporated herein by reference). The latter two amplificationmethods involve isothermal reactions based on isothermal transcription,which produce both single stranded RNA (ssRNA) and double stranded DNA(dsDNA) as the amplification products in a ratio of about 30 or 100 to1, respectively. Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.Ser. No. 09/854,317, each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 andU.S. patent application Ser. Nos. 09/916,135, 09/920,491, 09/910,292,and 10/013,598.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (3^(rd) Ed. ColdSpring Harbor, N.Y., 2002); Berger and Kimmel Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which areincorporated herein by reference

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred embodiments. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Patent application 60/364,731 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application60/364,731 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, e.g.Setubal and Meidanis et al., Introduction to Computational BiologyMethods (PWS Publishing Company, Boston, 1997); Salzberg, Searles,Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier,Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001).

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. patent application Ser. Nos. 10/063,559,60/349,546, 60/376,003, 60/394,574, 60/403,381.

(B.) Definitions

The term “admixture” refers to the phenomenon of gene flow betweenpopulations resulting from migration. Admixture can create linkagedisequilibrium (LD).

The term “allele’ as used herein is any one of a number of alternativeforms a given locus (position) on a chromosome. An allele may be used toindicate one form of a polymorphism, for example, a biallelic SNP mayhave possible alleles A and B. An allele may also be used to indicate aparticular combination of alleles of two or more SNPs in a given gene orchromosomal segment. The frequency of an allele in a population is thenumber of times that specific allele appears divided by the total numberof alleles of that locus.

The term “genome” as used herein is all the genetic material in thechromosomes of an organism. DNA derived from the genetic material in thechromosomes of a particular organism is genomic DNA. A genomic libraryis a collection of clones made from a set of randomly generatedoverlapping DNA fragments representing the entire genome of an organism.

The term “genotype” as used herein refers to the genetic information anindividual carries at one or more positions in the genome. A genotypemay refer to the information present at a single polymorphism, forexample, a single SNP. For example, if a SNP is biallelic and can beeither an A or a C then if an individual is homozygous for A at thatposition the genotype of the SNP is homozygous A or AA. Genotype mayalso refer to the information present at a plurality of polymorphicpositions.

The term “Hardy-Weinberg equilibrium” (HWE) as used herein refers to theprinciple that an allele that when homozygous leads to a disorder thatprevents the individual from reproducing does not disappear from thepopulation but remains present in a population in the undetectableheterozygous state at a constant allele frequency.

The term “linkage analysis” as used herein refers to a method of geneticanalysis in which data are collected from affected families, and regionsof the genome are identified that co-segregated with the disease in manyindependent families or over many generations of an extended pedigree. Adisease locus may be identified because it lies in a region of thegenome that is shared by all affected members of a pedigree.

The term “linkage disequilibrium” or sometimes referred to as “allelicassociation” as used herein refers to the preferential association of aparticular allele or genetic marker with a specific allele, or geneticmarker at a nearby chromosomal location more frequently than expected bychance for any particular allele frequency in the population. Forexample, if locus X has alleles A and B, which occur equally frequently,and linked locus Y has alleles C and D, which occur equally frequently,one would expect the combination AC to occur with a frequency of 0.25.If AC occurs more frequently, then alleles A and C are in linkagedisequilibrium. Linkage disequilibrium may result from natural selectionof certain combination of alleles or because an allele has beenintroduced into a population too recently to have reached equilibriumwith linked alleles. The genetic interval around a disease locus may benarrowed by detecting disequilibrium between nearby markers and thedisease locus. For additional information on linkage disequilibrium seeArdlie et al., Nat. Rev. Gen. 3:299-309, 2002.

The term “lod score” or “LOD” is the log of the odds ratio of theprobability of the data occurring under the specific hypothesis relativeto the null hypothesis. LOD=log [probability assuminglinkage/probability assuming no linkage].

Nucleic acids according to the present invention may include any polymeror oligomer of pyrimidine and purine bases, preferably cytosine,thymine, and uracil, and adenine and guanine, respectively. (See AlbertL. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)which is herein incorporated in its entirety for all purposes). Indeed,the present invention contemplates any deoxyribonucleotide,ribonucleotide or peptide nucleic acid component, and any chemicalvariants thereof, such as methylated, hydroxymethylated or glucosylatedforms of these bases, and the like. The polymers or oligomers may beheterogeneous or homogeneous in composition, and may be isolated fromnaturally occurring sources or may be artificially or syntheticallyproduced. In addition, the nucleic acids may be DNA or RNA, or a mixturethereof, and may exist permanently or transitionally in single-strandedor double-stranded form, including homoduplex, heteroduplex, and hybridstates.

An “oligonucleotide” or “polynucleotide” is a nucleic acid ranging fromat least 2, preferably at least 8, 15 or 20 nucleotides in length, butmay be up to 50, 100, 1000, or 5000 nucleotides long or a compound thatspecifically hybridizes to a polynucleotide. Polynucleotides of thepresent invention include sequences of deoxyribonucleic acid (DNA) orribonucleic acid (RNA) or mimetics thereof which may be isolated fromnatural sources, recombinantly produced or artificially synthesized. Afurther example of a polynucleotide of the present invention may be apeptide nucleic acid (PNA). (See U.S. Pat. No. 6,156,501 which is herebyincorporated by reference in its entirety.) The invention alsoencompasses situations in which there is a nontraditional base pairingsuch as Hoogsteen base pairing which has been identified in certain tRNAmolecules and postulated to exist in a triple helix. “Polynucleotide”and “oligonucleotide” are used interchangeably in this application.

The term “fragment,” “segment,” or “DNA segment” refers to a portion ofa larger DNA polynucleotide or DNA. A polynucleotide, for example, canbe broken up, or fragmented into, a plurality of segments. Variousmethods of fragmenting nucleic acid are well known in the art. Thesemethods may be, for example, either chemical or physical in nature.Chemical fragmentation may include partial degradation with a DNase;partial depurination with acid; the use of restriction enzymes;intron-encoded endonucleases; DNA-based cleavage methods, such astriplex and hybrid formation methods, that rely on the specifichybridization of a nucleic acid segment to localize a cleavage agent toa specific location in the nucleic acid molecule; or other enzymes orcompounds which cleave DNA at known or unknown locations (see, forexample, U.S. Ser. No. 09/358,664). Physical fragmentation methods mayinvolve subjecting the DNA to a high shear rate. High shear rates may beproduced, for example, by moving DNA through a chamber or channel withpits or spikes, or forcing the DNA sample through a restricted size flowpassage, e.g., an aperture having a cross sectional dimension in themicron or submicron scale. Other physical methods include sonication andnebulization. Combinations of physical and chemical fragmentationmethods may likewise be employed such as fragmentation by heat andion-mediated hydrolysis. See for example, Sambrook et al., “MolecularCloning: A Laboratory Manual,” 3^(rd) Ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001) (“Sambrook et al.) which isincorporated herein by reference for all purposes. These methods can beoptimized to digest a nucleic acid into fragments of a selected sizerange. Useful size ranges may be from 100, 200, 400, 700 or 1000 to 500,800, 1500, 2000, 4000 or 10,000 base pairs. However, larger size rangessuch as 4000, 10,000 or 20,000 to 10,000, 20,000 or 500,000 base pairsmay also be useful.

A number of methods disclosed herein require the use of restrictionenzymes to fragment the nucleic acid sample. In general, a restrictionenzyme recognizes a specific nucleotide sequence of four to eightnucleotides and cuts the DNA at a site within or a specific distancefrom the recognition sequence. For example, the restriction enzyme EcoRIrecognizes the sequence GAATTC and will cut a DNA molecule between the Gand the first A. The length of the recognition sequence is roughlyproportional to the frequency of occurrence of the site in the genome. Asimplistic theoretical estimate is that a six base pair recognitionsequence will occur once in every 4096 (4⁶) base pairs while a four basepair recognition sequence will occur once every 256 (4⁴) base pairs. Insilico digestions of sequences from the Human Genome Project show thatthe actual occurrences may be more or less frequent, depending on thesequence of the restriction site. Because the restriction sites arerare, the appearance of shorter restriction fragments, for example thoseless than 1000 base pairs, is much less frequent than the appearance oflonger fragments. Many different restriction enzymes are known andappropriate restriction enzymes can be selected for a desired result.(For a description of many restriction enzymes and their recognitionsites and optimal buffer conditions see, New England BioLabs Catalogwhich is herein incorporated by reference in its entirety for allpurposes).

Type-IIs endonucleases are a class of endonucleases that, like otherendonucleases, recognize specific sequences of nucleotide base pairswithin a double stranded polynucleotide sequence. Upon recognizing thatsequence, the endonuclease will cleave the polynucleotide sequence,generally leaving an overhang of one strand of the sequence, or “stickyend.” The Type-IIs endonucleases are unique because they generally donot require palindromic recognition sequences and they generally cleaveoutside of their recognition sites. The recognition sequence is oftennon-palindromic, and the cleavage occurs outside of the recognitionsite.

Type-IIs endonucleases are generally commercially available and are wellknown in the art. Specific Type-IIs endonucleases which are useful inthe present invention include, e.g., BbvI, BceAI, BfuAI, EarI, AlwI,BbsI, BsaI, BsmAI, BsmBI, BspMI, HgaI, SapI, SfaNI, BsmFI, FokI, andPleI. Other Type-IIs endonucleases that may be useful in the presentinvention may be found, for example, in the New England Biolabscatalogue. In some embodiments Type-IIs enzymes that generate a recessed3′ end are particularly useful.

“Adaptor sequences” or “adaptors” are generally oligonucleotides of atleast 5, 10, 15 or 20 bases and preferably no more than 50 or 60 basesin length; however, they may be even longer, up to 100 or 200 bases.Adaptor sequences may be synthesized using any methods known to those ofskill in the art. For the purposes of this invention they may, asoptions, comprise primer binding sites, recognition sites forendonucleases, common sequences and promoters. The adaptor may beentirely or substantially double stranded. A double stranded adaptor maycomprise two oligonucleotides that are at least partially complementary.The adaptor may be phosphorylated or unphosphorylated on one or bothstrands. Adaptors may be more efficiently ligated to fragments if theycomprise a substantially double stranded region and a short singlestranded region which is complementary to the single stranded regioncreated by digestion with a restriction enzyme. For example, when DNA isdigested with the restriction enzyme EcoRI the resulting double strandedfragments are flanked at either end by the single stranded overhang5′-AATT-3′, an adaptor that carries a single stranded overhang5′-AATT-3′ will hybridize to the fragment through complementaritybetween the overhanging regions. This “sticky end” hybridization of theadaptor to the fragment may facilitate ligation of the adaptor to thefragment but blunt ended ligation is also possible. Blunt ends can beconverted to sticky ends using the exonuclease activity of the Klenowfragment. For example when DNA is digested with PvuII the blunt ends canbe converted to a two base pair overhang by incubating the fragmentswith Klenow in the presence of dTTP and dCTP. Overhangs may also beconverted to blunt ends by filling in an overhang or removing anoverhang.

An adaptor may be ligated to one or both strands of the fragmented DNA.In some embodiments a double stranded adaptor is used but only onestrand is ligated to the fragments. Ligation of one strand of an adaptormay be selectively blocked. Any known method to block ligation of onestrand may be employed. For example, one strand of the adaptor can bedesigned to introduce a gap of one or more nucleotides between the 5′end of that strand of the adaptor and the 3′ end of the target nucleicacid. Adaptors can be designed specifically to be ligated to the terminiproduced by restriction enzymes and to introduce gaps or nicks. Forexample, if the target is an EcoRI digested fragment an adaptor with a5′ overhang of TTA could be ligated to the AATT overhang left by EcoRIto introduce a single nucleotide gap between the adaptor and the 3′ endof the fragment. Phosphorylation and kinasing can also be used toselectively block ligation of the adaptor to the 3′ end of the targetmolecule. Absence of a phosphate from the 5′ end of an adaptor willblock ligation of that 5′ end to an available 3′OH. For additionaladaptor methods for selectively blocking ligation see U.S. Pat. No.6,197,557 and U.S. Ser. No. 09/910,292 which are incorporated byreference herein in their entirety for all purposes.

Adaptors may also incorporate modified nucleotides that modify theproperties of the adaptor sequence. For example, phosphorothioate groupsmay be incorporated in one of the adaptor strands. A phosphorothioategroup is a modified phosphate group with one of the oxygen atomsreplaced by a sulfur atom. In a phosphorothioated oligo (often called an“S-Oligo”), some or all of the internucleotide phosphate groups arereplaced by phosphorothioate groups. The modified backbone of an S-Oligois resistant to the action of most exonucleases and endonucleases.Phosphorothioates may be incorporated between all residues of an adaptorstrand, or at specified locations within a sequence. A useful option isto sulfurize only the last few residues at each end of the oligo. Thisresults in an oligo that is resistant to exonucleases, but has a naturalDNA center.

Methods of ligation will be known to those of skill in the art and aredescribed, for example in Sambrook et at. (2001) and the New EnglandBioLabs catalog both of which are incorporated herein by reference forall purposes. Methods include using T4 DNA Ligase which catalyzes theformation of a phosphodiester bond between juxtaposed 5′ phosphate and3′ hydroxyl termini in duplex DNA or RNA with blunt and sticky ends; TaqDNA Ligase which catalyzes the formation of a phosphodiester bondbetween juxtaposed 5′ phosphate and 3′ hydroxyl termini of two adjacentoligonucleotides which are hybridized to a complementary target DNA; E.coli DNA ligase which catalyzes the formation of a phosphodiester bondbetween juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNAcontaining cohesive ends; and T4 RNA ligase which catalyzes ligation ofa 5′ phosphoryl-terminated nucleic acid donor to a 3′hydroxyl-terminated nucleic acid acceptor through the formation of a3′→5′ phosphodiester bond, substrates include single-stranded RNA andDNA as well as dinucleoside pyrophosphates; or any other methodsdescribed in the art.

When a fragment has been digested on both ends with the same enzyme ortwo enzymes that leave the same overhang, the same adaptor may beligated to both ends. Digestion with two or more enzymes can be used toselectively ligate separate adaptors to either end of a restrictionfragment. For example, if a fragment is the result of digestion withEcoRI at one end and BamHI at the other end, the overhangs will be5′-AATT-3′ and 5′GATC-3′, respectively. An adaptor with an overhang ofAATT will be preferentially ligated to one end while an adaptor with anoverhang of GATC will be preferentially ligated to the second end.

A genome is all the genetic material of an organism. In some instances,the term genome may refer to the chromosomal DNA. Genome may bemultichromosomal such that the DNA is cellularly distributed among aplurality of individual chromosomes. For example, in human there are 22pairs of chromosomes plus a gender associated XX or XY pair. DNA derivedfrom the genetic material in the chromosomes of a particular organism isgenomic DNA. The term genome may also refer to genetic materials fromorganisms that do not have chromosomal structure. In addition, the termgenome may refer to mitochondria DNA. A genomic library is a collectionof DNA fragments representing the whole or a portion of a genome.Frequently, a genomic library is a collection of clones made from a setof randomly generated, sometimes overlapping DNA fragments representingthe entire genome or a portion of the genome of an organism.

The term “chromosome” refers to the heredity-bearing gene carrier of aliving cell which is derived from chromatin and which comprises DNA andprotein components (especially histones). The conventionalinternationally recognized individual human genome chromosome numberingsystem is employed herein. The size of an individual chromosome can varyfrom one type to another with a given multi-chromosomal genome and fromone genome to another. In the case of the human genome, the entire DNAmass of a given chromosome is usually greater than about 100,000,000 bp.For example, the size of the entire human genome is about 3×10⁹ bp. Thelargest chromosome, chromosome no. 1, contains about 2.4×10⁸ by whilethe smallest chromosome, chromosome no. 22, contains about 5.3×10⁷ bp.

A chromosomal region is a portion of a chromosome. The actual physicalsize or extent of any individual chromosomal region can vary greatly.The term region is not necessarily definitive of a particular one ormore genes because a region need not take into specific account theparticular coding segments (exons) of an individual gene.

An allele refers to one specific form of a genetic sequence (such as agene) within a cell, an individual or within a population, the specificform differing from other forms of the same gene in the sequence of atleast one, and frequently more than one, variant sites within thesequence of the gene. The sequences at these variant sites that differbetween different alleles are termed “variances”, “polymorphisms”, or“mutations”. At each autosomal specific chromosomal location or “locus”an individual possesses two alleles, one inherited from one parent andone from the other parent, for example one from the mother and one fromthe father. An individual is “heterozygous” at a locus if it has twodifferent alleles at that locus. An individual is “homozygous” at alocus if it has two identical alleles at that locus.

Capture probes are oligonucleotides that have a 5′ common primingsequence and a 3′ locus or target specific region or primer. The locusor target specific region is designed to hybridize near a region ofnucleic acid that includes a region of interest, for example, near apolymorphism, so that the locus or target specific region of the captureprobe can be used as a primer and be extended through the region ofinterest to make a copy of the region of interest. The common primingsequence in the capture probe may be used as a priming site insubsequent rounds of amplification using a common primer or a limitednumber of common primers. The same common priming sequence may bepresent in many or all or the capture probes in a collection of captureprobes so that extended capture probes can be amplified with a commonsequence primer, for example T7 promoter primer. Capture probes may alsocomprise other sequences, for example, tag sequences that are unique fordifferent species of capture probes, and endonuclease recognition sites.In some embodiments the capture probe is designed to hybridize upstreamof a polymorphic position so that the capture probe can be extendedthrough the polymorphic position, thus incorporating a copy of thepolymorphism into the extended capture probe. In some embodiments thetarget sample is fragmented prior to extension of the capture probes andextension terminates at the end of the fragments. The fragments may ormay not be ligated to adaptors before capture probe extension. If thefragments are ligated to an adaptor sequence, the extended capture probewill be extended through the adaptor and will terminate in the adaptorsequence.

A degenerate capture probe is a capture probe that has a degeneratelocus or target specific region or primer. The locus specific region isthe region of the capture probe that is complementary to the targetupstream of the polymorphism. The locus specific region is the 3′ regionof the capture probe so that it can hybridize to the target upstream ofthe polymorphism and be extended through the polymorphic site, thusincorporating the polymorphic base into the extended capture probe. Adegenerate capture probe is synthesized with some positions beingvariable and some positions being fixed. For example, the locus specificregion of a degenerate capture probe may be, for example,5′-ACNNGTNNNNAATT-3′ (SEQ ID No. 1). Positions 1, 2, 5, 6, and 11-14 arefixed and positions 3, 4, and 7-10 are variable and can be A, G, C or T.

A degenerate priming site is a site that is complementary to onepossible species of a degenerate primer. For example, if the degenerateprimer sequence is 5′-ACNNGTNNNNAATT-3′ (SEQ ID NO. 1) then degeneratepriming sites would include, for example, 5′-AATTtaccACgtGT-3′ (SEQ IDNO. 2) and 5′-AATTgcaaACccGT-3′ (SEQ ID NO. 3). In these examples thelower case letters represent positions that may vary from probe to probeand the upper case letters are positions that are constant in all probeswith that degenerate sequence. Each degenerate primer sequencecorresponds to many different sequences all sharing the same constantnucleotides at constant positions and representing all possiblevariations of the N positions.

A tag or tag sequence is a selected nucleic acid with a specifiednucleic acid sequence. A tag probe has a region that is complementary toa selected tag. A set of tags or a collection of tags is a collection ofspecified nucleic acids that may be of similar length and similarhybridization properties, for example similar T_(m). The tags in acollection of tags bind to tag probes with minimal cross hybridizationso that a single species of tag in the tag set accounts for the majorityof tags which bind to a given tag probe species under hybridizationconditions. For additional description of tags and tag probes andmethods of selecting tags and tag probes see U.S. Ser. No. 08/626,285and EP/0799897, each of which is incorporated herein by reference intheir entirety.

A collection of capture probes may be designed to interrogate acollection of target sequences. The collection would comprise at leastone capture probe for each target sequence to be amplified. There may bemultiple different capture probes for a single target sequence in acollection of capture probes, for example, there may be a capture probethat hybridizes to one strand of the target sequence and a capture probethat hybridizes to the opposite strand of the target sequence, these maybe referred to as a forward locus or target specific primer and areverse locus or target specific primer. There also may be two or morecapture probes that hybridize at different locations downstream of thetarget sequence.

A collection of capture probes may be used to amplify a subset of agenome. The collection of capture probes may be initially used togenerate a copy of the target sequences in the genomic sample and thenthe copies may be amplified using common primers. The amplification maybe done simultaneously in the same reaction and often in the same tube.

The term “target sequence”, “target nucleic acid” or “target” refers toa nucleic acid of interest. The target sequence may or may not be ofbiological significance. As non-limiting examples, target sequences mayinclude regions of genomic DNA which are believed to contain apolymorphism. The number of sequences to be interrogated can vary, butpreferably are from about 1000, 2,000, 5,000, 10,000, 20,000 or 100,000to 5000, 10,000, 100,000, 1,000,000 or 3,000,000 target sequences.

An “array” comprises a support, preferably solid, with nucleic acidprobes attached to the support. Preferred arrays typically comprise aplurality of different nucleic acid probes that are coupled to a surfaceof a substrate in different, known locations. These arrays, alsodescribed as “microarrays” or colloquially “chips” have been generallydescribed in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934,5,744,305, 5,677,195, 5,800,992, 6,040,193, 5,424,186 and Fodor et al.,Science, 251:767-777 (1991). Each of which is incorporated by referencein its entirety for all purposes.

Arrays may generally be produced using a variety of techniques, such asmechanical synthesis methods or light directed synthesis methods thatincorporate a combination of photolithographic methods and solid phasesynthesis methods. Techniques for the synthesis of these arrays usingmechanical synthesis methods are described in, e.g., U.S. Pat. Nos.5,384,261, and 6,040,193, which are incorporated herein by reference intheir entirety for all purposes. Although a planar array surface ispreferred, the array may be fabricated on a surface of virtually anyshape or even a multiplicity of surfaces. Arrays may be nucleic acids onbeads, gels, polymeric surfaces, fibers such as fiber optics, glass orany other appropriate substrate. (See U.S. Pat. Nos. 5,770,358,5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are herebyincorporated by reference in their entirety for all purposes.)

A genotyping array comprises probes that are specific for one allele ofa polymorphism. Genotyping arrays are described, for example, in U.S.patent application Ser. Nos. 10/264,945 and 10/442,021 and U.S.Provisional patent applications Nos. 60/470,475 filed May 14, 2003,60/483,050 filed Jun. 27, 2003 and 60/417,190 filed Oct. 8, 2002, eachof which is incorporated herein by reference in its entirety.

Arrays may be packaged in such a manner as to allow for diagnostic useor can be an all-inclusive device; e.g., U.S. Pat. Nos. 5,856,174 and5,922,591 incorporated in their entirety by reference for all purposes.

Preferred arrays are commercially available from Affymetrix under thebrand name GeneChip® and are directed to a variety of purposes,including genotyping and gene expression monitoring for a variety ofeukaryotic and prokaryotic species. (See Affymetrix Inc., Santa Claraand their website at affymetrix.com.) A genotyping array such as theHuman Mapping Array 10K Xba 131 may be used to determine the genotype ofa collection of SNPs by hybridization. The array contains probes thatare specific for each possible allele for a collection of SNPs.Fragments that carry the SNPs are amplified, labeled and hybridized tothe array. The presence of a fragment is determined by the hybridizationpattern. For additional description of a genotyping array see U.S.provisional patent application No. 60/417,190 filed Oct. 8, 2002.

Combinatorial chemistry may be used for the parallel synthesis ofdiscreet compounds, for example, oligonucleotides of different sequenceon a solid support. See, for example, U.S. Pat. Nos. 5,412,087,5,424,186, 5,445,934 and 6,040,193 which are each incorporated herein byreference. Many different compounds may be synthesized. The compoundsmay be oligonucleotides which may be synthesized on a solid support sothat each discreet compound or oligonucleotide is localized to aspecific region, or feature, of the array which may be predefined. Insome embodiments there may be overlap between regions. In someembodiments a plurality of different oligonucleotides are generated,each sharing a core set of bases but differing at some positions. Forexample, each feature of the array may be of the sequence5′-GAATNNCNG-3′ and within each discreet feature the N's will be thesame, for example one feature may be 5′-GAATcgCtG-3′ while anotherfeature may be 5′-GAATttCgG-3′. The core bases are GAAT-C-G. Thesynthesis of many different probes may be accomplished in this mannerwith increase efficiency and decreased cost because the majority ofprobes of the array have the same core set of bases and addition ofthose bases may be done en masse without the use of feature specificphotolithography, i.e. all features may be activated simultaneously forthose positions. In some embodiments there are control oligonucleotideson the array that may or may not share the common core bases.

Hybridization probes are oligonucleotides capable of binding in abase-specific manner to a complementary strand of nucleic acid. Suchprobes include peptide nucleic acids, as described in Nielsen et al.,Science 254, 1497-1500 (1991), and other nucleic acid analogs andnucleic acid mimetics. See U.S. Pat. No. 6,156,501.

The term hybridization refers to the process in which twosingle-stranded polynucleotides bind non-covalently to form adouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting double-stranded polynucleotide isa “hybrid.” The hybrid may have double-stranded regions and singlestranded regions.

Hybridizations are usually performed under stringent conditions, forexample, at a salt concentration of no more than 1 M and a temperatureof at least 25° C. For example, conditions of 5×SSPE (750 mM NaCl, 50 mMNaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. aresuitable for allele-specific probe hybridizations. For stringentconditions, see, for example, Sambrook et al., (2001) which is herebyincorporated by reference in its entirety for all purposes above.

An individual is not limited to a human being, but may also includeother organisms including but not limited to mammals, plants, bacteriaor cells derived from any of the above.

(C) Multiplexed Locus Specific Genotyping

Generally, the invention provides methods for highly multiplexed locusspecific amplification of nucleic acids and methods for analysis of theamplified products. In preferred embodiments the amplified targetsprepared using Multiplexed Anchored Runoff Amplification (MARA) areanalyzed to determine the genotype of SNPs. In some embodiments theinvention combines the use of capture probes that comprise a commonsequence and a locus-specific region with adaptor-modified samplenucleic acid; the adaptor comprises a second common sequence. Thecapture probes are extended to produce copies of the sample DNA thatcontain common priming sequences flanking the target sequence. Thecopies are amplified with a generic set of primers that recognize thecommon sequences. The amplified product may be analyzed by hybridizationto an array of probes.

In one embodiment the steps of the invention comprise: designing andsynthesizing capture probes; digesting a nucleic acid sample; ligatingadaptors to the fragmented sample; mixing the fragments and the captureprobes under conditions that will allow hybridization of the fragmentsand the capture probes; extending the capture probes in the presence ofdNTPs and polymerase; amplifying the extended capture probes usingprimers to the common sequences in the capture probe and the adaptor;and detecting the presence or absence of target sequences of interest.In a preferred embodiment allele discrimination is achieved byhybridization to high density DNA oligonucleotide arrays.

One embodiment of the methods is illustrated in FIG. 1. Capture probesare designed with a locus specific region (LS1_(F) and LS1_(R)) thathybridizes near a target sequence of interest and a common sequence (A1)that is 5′ of the locus specific region. The common priming site may bepresent in a plurality of capture probes so that a primer to A1 may beused for amplification of a plurality of different targets in subsequentsteps. The capture probes may be attached to a solid support so thatthey have a free 3′ end or the extension may be done in solution. Aplurality of a single species of capture probe may be synthesized at adiscreet location on an array and may form a discrete feature of anarray. Each feature of the array may contain a different species oflocus specific capture probe at a known or determinable location.

Genomic DNA is fragmented and adaptors comprising a second commonsequence (A2) are ligated to the fragments. The adaptor-ligatedfragments are then mixed with the capture probes under conditions thatallow hybridization of the fragments to the capture probes on the array.The capture probes are then extended using the adaptor-ligated fragmentsas template. The extension product has a common sequence, A1, near its5′ end and a second common sequence A2 near its 3′ end. These commonsequences flank a region of interest. The capture probes are thenreleased from the array and extended capture probes are amplified by PCRusing primers to the common sequences A1 and A2. The amplified productmay then be analyzed by, for example, hybridization to an array.Information about the region of interest can be determined by analysisof the hybridization pattern.

A second embodiment of the methods is illustrated in FIG. 2. Captureprobes are designed with a locus specific region (LS1 or LS2) and acommon sequence (A1) as in FIG. 1. In this embodiment the capture probesfurther comprise a tag sequence that is unique for each species ofcapture probe designed. (For a description of tags and tag probes, see,U.S. Ser. No. 08/626,285.) The capture probes are attached to the arraythrough hybridization of the tag sequence to a substantiallycomplementary tag probe sequence that is attached to the array. The tagprobes may be attached to the array in discrete locations. Differentspecies of tag probes are present at different discrete, spatiallyaddressable locations. Adaptor-ligated genomic DNA is hybridized to thearray so that the capture probes hybridize to target sequences in thesample. The capture probes are extended as in FIG. 1 to incorporate thetarget sequence and common sequence A2. The extended capture probes arereleased and amplified using primers A1 and A2. The amplified productmay then be analyzed by, for example, hybridization to an array.Information about the region of interest can be determined by analysisof the hybridization pattern. The amplified sample may be analyzed byany method known in the art, for example, MALDI-TOF mass spec, capillaryelectrophoresis, OLA, dynamic allele specific hybridization (DASH) orTaqMan® (Applied Biosystems, Foster City, Calif.). For other methods ofgenotyping analyses see Syvanen, Nature Rev. Gen. 2:930-942 (2001) whichis herein incorporated by reference in its entirety.

In some embodiments the capture probes are attached to a solid supportprior to hybridization and hybridization takes place while the captureprobes are attached to the solid support. In some embodiments thecapture probes are synthesized on a solid support. Any suitable solidsupport known in the art may be used, for example, arrays, beads,microparticles, microtitre dishes and gels may be used. In someembodiments the capture probes are synthesized on an array in a 5′ to 3′direction.

In some embodiments hybridization and extension of capture probes aredone while the capture probes are attached to a solid support. Followingextension of the capture probes nucleic acids that are not covalentlyattached to the solid support may be washed away. In some embodimentsthe extended capture probes are released from the solid support prior toamplification. In another embodiment amplification takes place while theextended capture probes are attached to the solid support. The extendedcapture probes may be released from the solid support by, for example,using a reversible linker or an enzymatic release, such as anendonuclease or by a change in conditions that results in disruption ofan interaction between the capture probe and the solid support, forexample, when capture probes are associated with the solid supportthrough base pairing between a tag in the capture probe and a tag probeon the solid support, disruption of the base pairing interactionreleases the capture probes from the solid support. Enzymatic methodsinclude, for example, use of uracil DNA glycosylase (UDG) or (UNG). UNGcatalyzes the hydrolysis of DNA that contains deoxyuridine at the sitethe uridine is incorporated. Incorporation of one or more uridines inthe capture probe followed by treatment with UNG will result in releaseof the capture probe from the solid support. A thermolabile UNG may alsobe used.

In preferred embodiments a collection of target sequences is analyzed. Aplurality of capture probes is designed for a plurality of targetsequences. Pools of more than 250, more than 750, more than 1,000 ormore than 4,000 different capture probes may be simultaneously extendedin a multiplexed reaction. In some embodiments target sequences containor are predicted to contain a polymorphism, for example, a SNP. Thepolymorphism may be, for example, near a gene that is a candidate markerfor a phenotype, useful for diagnosis or a disorder or for carrierscreening or the polymorphism may define a haplotype block (see, Daly etal. Nat Genet. 29:229-32 (2001), and Rioux et al. Nat Genet. 29:223-8(2001) and U.S. patent application Ser. No. 10/213,272, each of which isincorporated herein by reference in its entirety). A collection ofcapture probes may be designed so that capture probes hybridize near apolymorphism, for example, within 1, 5, 10, or 100 to 5, 10, 100, 1000,10,000 or 100,000 bases from the polymorphism. The capture probeshybridize to one strand of the target sequence and can be extendedthrough the polymorphic site or region so that the extension productcomprises a copy of the polymorphic region.

Many amplification methods are most efficient at amplification ofsmaller fragments. For example, PCR most efficiently amplifies fragmentsthat are smaller than about 2 kb (see, Saiki et al. 1988). In oneembodiment capture probes and fragmentation conditions are selected forefficient amplification of a selected collection of target sequences.The size of the amplified fragments is dependent on where the targetspecific region of the capture probe hybridizes to the target sequenceand the 5′ end of the fragment strand that the capture probe ishybridized to. In some embodiments of the present methods capture probesand fragmentation methods are designed so that the target sequence ofinterest can be amplified as a fragment that is, for example, less than20,000, 2,000, 1,000, 800, 500, 400, 200 or 100 base pairs long.Multiplex PCR methods and methods to improve PCR amplification have beenshown. See for example, Edwards and Gibbs PCR Methods Appl 3: S65-75(1994), Henegariu, et al. Biotechniques 23: 504-511 (1997), Shuber etal. Genome Res 5: 488-493 (1995), Broude, et al. Antisense Nucleic AcidDrug Dev 11: 327-332 (2001), Broude, et al. Proc Natl Acad Sci USA 98:206-211 (2001) and Brownie, et al. Nucleic Acids Res 25: 3235-3241(1997).

For multiplexed amplification capture probes can be designed so that the3′ end of the target specific region hybridizes to the base that isimmediately 3′ of a position to be interrogated in the target sequence.For example, if the sequence to be interrogated is a polymorphism andthe sequence is 5′-GCTXATCGG-3′, where X is the polymorphic position,the target specific region of the capture probe may have the sequence5′-CCGAT-3′. When the sample is fragmented with site specificrestriction enzymes the length of the fragments will also depend on theposition of the nearest recognition site for the enzyme or enzymes usedfor fragmentation. A collection of target sequences may be selectedbased on proximity to restriction sites. In some embodiments targetsequences are selected for amplification and analysis based on thepresence of a sequence of interest, such as a SNP, and proximity to acleavage site for a selected restriction enzyme. For example, SNPs thatare within 200, 500, 800, 1,000, 1,500, 2,000 or 20,000 base pairs ofeither a restriction site, such as, for example, an EcoRI site, a BglIsite, an XbaI site, a NotI site or any other restriction enzyme site maybe selected to be target sequences in a collection of target sequences.Preferably the sequence to be interrogated, for example the polymorphicposition, is between the target specific sequence of the capture probeand the downstream restriction site.

In another embodiment a fragmentation method that randomly cleaves thesample into fragments that are 30,100, 200, 500 or 1,000 to 100, 200,500, 1,000 or 2,500 base pairs on average may be used. Fragmentation mayoccur before or after extension of capture probes. When fragmentation isperformed prior to extension of capture probes an adaptor may be ligatedto the fragments. Fragments may be blunt ended and a blunt end adaptormay be ligated. When the capture probe is extended the adaptor sequencewill be incorporated. If fragmentation is performed after capture probeextension an adaptor may be ligated to the end of the extended captureprobe to incorporate a primer binding site.

In another embodiment, illustrated in FIG. 3, the capture probes are insolution and hybridization and extension take place in solution. In thisembodiment the nucleic acid sample is fragmented and adaptor containingcommon sequences A2 and A3 is ligated to the fragments. In someembodiments one strand of the adaptor, the strand that is ligated to the3′ end of the fragment strands lacks common sequence A2 and is blockedfrom extension at the 3′ end. Ligation of the blocked adaptor strand tothe 3′ end of the fragment strands prevents the fragments from beingextended to incorporate A2 at both ends, thus preventing amplificationof the fragments by primer A2 in the subsequent PCR amplification step.Capture probes with locus specific regions and common sequence A1 aremixed with the adaptor-ligated fragments under conditions that allowhybridization of the capture probes to the adaptor ligated fragments.The capture probes are extended in the presence of polymerase and dNTPs.In some embodiments the extended capture probes are positively selectedto generate a sample that is enriched for extended capture probes. Inanother embodiment extended capture probes are enriched by depletingnon-extended products.

MARA primers with Degenerate Locus Specific Region. Many of thedisclosed methods, as well as many other genotyping methods, utilizeprimers comprising locus specific regions. As the number ofpolymorphisms to be genotyped increases so does the number of locusspecific primers that must be synthesized. Synthesis of large numbers oflocus specific primers by standard methods can be cost prohibitive.Handling large numbers of individually aliquoted locus specific primers,for example in 96 well plates, may also be difficult and errors may beintroduced, for example putting the wrong primer in the wrong well ormixing primers in a well. Synthesis of locus specific primers directlyon a solid support may be used to simplify and reduce the cost ofsynthesizing large numbers of locus specific oligonucleotides.Combinatorial synthesis of locus specific primers, for example, MARAcapture probes, with degenerate positions may also improve efficiencyand reduce the cost of locus specific genotyping applications. Thedegenerate capture probes may be synthesized by any method known, forexample on a solid support such as on an array or on beads. Methods forcombinatorial synthesis are described in U.S. Pat. No. 5,541,061.Degenerate-oligonucleotide-primed PCR methods have been described inTelenius et al. Genomics 13:718-725 (1992).

MARA capture probes may be designed so that the locus specific region ofa plurality of primers share some common sequence features. In oneembodiment polymorphisms are selected for genotyping based on thepresence of common sequence motifs. For example, polymorphisms may beselected that are within 100, 200, 500, or 1000 base pairs of a selectedcommon sequence of 4, 5, 6, 8 or 10 bases, the selected sequence may be,for example, a restriction enzyme recognition site. The polymorphismsare then analyzed to identify a subset that also shares a consensussequence immediately upstream of the selected common sequence. Theconsensus sequence may be for example a stretch of 8-20 bases. Some ofthe bases are identical for each polymorphism in the subset and some ofthe bases vary from one polymorphism to the next. An example of this isillustrated in FIG. 4. The three loci share a consensus sequenceupstream of a polymorphism. The common sequence shared by each is ATTAAand the similar sequence is GANNN immediately upstream of the ATTAA. Thesame partially degenerate primer with locus specific sequence 5′NNGANNNATTAA 3′ (SEQ ID NO. 4), can be hybridized to each of the threeloci and extended.

In a preferred embodiment the locus specific region of at least some ofthe capture probes contains degenerate positions. An oligonucleotidepool may be synthesized so that some positions are a single fixednucleotide and some positions can be one of two or more differentnucleotides. For example the sequence of the locus specific region ofsome of the oligonucleotides may be 5′-NNGANNNATTAA-3′, where N can beany nucleotide, including G, C, A and T. There are 4⁵ or 1024 differentsequences possible for this oligonucleotide if N is either G, C, A or T.Each of the 1024 sequences may occur at many places in the genome.Assuming random distribution of sequences any given 12 mer should occurapproximately 180 times in the 3 billion base pair human genome (4¹² is˜1.7×10⁷ and 3×10⁹/1.7×10⁷ is ˜180). A plurality of SNPs may be assayedusing a single degenerate pool of oligonucleotides. If each of the 1024sites is present 180 times in the genome, a single degenerateoligonucleotide synthesis could hybridize and serve as a primer at180,000 different sites (1024×180 is 184,320). A plurality of thecapture probes share common sequences separated by variable sequences.For example, in FIG. 4 the locus 1, 2 and 3 priming sites share thesequence NNCTNNNTAATT where the underlined nucleotides are commonbetween each of the three sites and the nucleotides indicated by N arevariable. An oligonucleotide that is degenerate at each of the Npositions and has the common sequences could be used to amplify each ofthe three loci.

In one embodiment combinatorial chemistry is used to synthesize a poolof capture probes. The use of combinatorial chemistry allows a largenumber of oligonucleotides of different sequence to be synthesized enmasse at a reduced cost. In one embodiment some positions in theoligonucleotides will be defined and some will be variable. In someembodiments the capture probes are synthesized on an array and somepositions of at least some of the oligonucleotides are variable. Theinclusion of a variable position allows for the synthesis of multipledifferent oligonucleotides at a single feature of the array. Captureprobes may be synthesized on a solid support at distinct positions orfeatures using, for example photolithography or printing methods. Thevariable positions may be at the same location in all of the features orat different locations that vary from feature to feature. For example,all of the probes of the array may have variable positions at positions5, 10, 11, and 15-20 or some of the features may have variable positionsat these positions and some may have variable positions at otherpositions that are different from these, for example at 5, 12-16 and 22.The probes may be synthesized on an array as disclosed in U.S. Pat. No.5,412,087 and the method of synthesis may be designed to minimize stepsand cost by, for example, the methods disclosed in U.S. Pat. No.5,593,839.

Methods of combinatorial synthesis of polymer libraries have beendescribed in U.S. Pat. No. 5,541,061. A large number of differentsequence probes may be synthesized in a series of pooled and separatesynthesis steps so that a large number of different polymers may besynthesized in a reduced number of steps. Pools of probes can besynthesized so that all of the probes in a given pool share commonsequence elements and vary at similar positions. The pools of probes canbe efficiently synthesized in a reduced number of steps by using pooledand unpooled synthesis steps.

In one embodiment a collection of SNPs is first selected for genotyping.Capture probes are then designed for the collection of SNPs. Each SNP tobe genotyped is analyzed to identify a common sequence near the SNP, forexample, a restriction enzyme recognition site. In one embodiment thecommon sequence is within 1000 or 2000 base pairs of the polymorphism.The sequence surrounding the common sequence is then analyzed andcompared to the sequence surrounding that common sequence in other SNPsof the collection to identify a subset of SNPs that have additionalcommon sequences surrounding the first common sequence (see FIGS. 6A and6B). Degenerate primers are designed that have constant sequences atpositions that are common among the SNPs in the subset. These steps maybe repeated for other subsets so that a single degenerate capture probecan be used for each identified subset of SNPs.

In another embodiment a pool of locus specific capture probes issynthesized using combinatorial chemistry methods as disclosed in U.S.Pat. No. 5,541,061. Capture probes are synthesized in pools using aseries of separate and pooled monomer addition steps. The use ofseparate and pooled steps allows a larger number of different locusspecific probes to be synthesized using fewer steps. For example, FIG.6A shows a schematic of a combinatorial synthesis process. In the firststep monomers A and B are added separately, then they are pooled and Cis added, the sample is separated and D and E are added, then pooled andF is added and finally G and H are added to separate pools. The finalresult is two pools each with 4 different polymers, (ACDFG, ACEFG,BCDFG, BCEFG and ACDFH., ACEFH, BCDFH, BCEFH). This requires 8 separateaddition steps. FIG. 6B shows that if each monomer was added separatelyit would require 16 steps to synthesize the same 16 polymers. Aplurality of capture probes may be designed to interrogate a pluralityof SNPs and the probes may be synthesized in pools using a combinatorialsynthesis strategy.

In one embodiment a collection of SNPs to be genotyped is identified anda collection of capture probes is designed so that a minimum number ofprobes may be synthesized to genotype the collection of SNPs. Some ofthe capture probes may be synthesized to genotype a specific SNP andsome capture probes may be synthesized to include degenerate positionsso that a single synthesis may be used to generate multiple differentcapture probes that are each specific for a SNP. In some embodiments thecapture probes also have a common sequence, such as a T7 promotersequence, that may be used as a primer binding site so that a commonprimer may be used to amplify extension products. For example, the poolof capture probes may comprise5′TAATACGACTCACTATAGGGAGAACNNGTNNNNAATT-3′ (SEQ ID NO. 5). The pool ofdegenerate capture probes will bind to many sites within the genome.

In one embodiment the degenerate capture probes are hybridized to thetargets and extended using the target as template. The extended probesmay be digested with a restriction enzyme that may cut within 200, 500or 1000 bp of the capture probe binding site but not between thepolymorphism to be detected and the capture probe binding site.Digestion may be done before extension so that the double strandedtarget is digested or after extension so that the extended captureprobe-target duplex is digested. After digestion, adaptors may beligated to the overhang. The adaptor may have a common priming sequence.After extension and adaptor ligation the products may be amplified witha T7 primer and a primer complementary to the ligated adaptor.

In one embodiment a collection of degenerate locus specific primers isdesigned to hybridize near a polymorphism so that the polymorphism isbetween the site of primer binding and a restriction site. In oneembodiment polymorphisms and priming sites are selected so that thedistance between the restriction site and the primer binding site iswithin 200, 500, 1000 or 2000 base pairs. In some embodiments thedistance is less than about 5,000 or less than about 10,000 base pairs.In one embodiment each degenerate locus specific primer has a commonpriming sequence at the 5′ end and a degenerate sequence near the 3′end. In one embodiment the degenerate locus specific primer also has atag sequence which may be between the common priming sequence and thedegenerate sequence.

In one embodiment polymorphisms to be analyzed are selected to have thefollowing criteria: the polymorphism is located near a potential primingsite (the priming site is where the capture probe hybridizes) for aselected degenerate capture probe, for example, within 500, 1000, 2000,5000 or 10,000 base pairs of the priming site; a selected restrictionsite is located near the polymorphism, for example, within 500, 1000,2000, 5000 or 10,000 base pairs of the polymorphism; and thepolymorphism is between the priming site and the restriction site. Inone embodiment a plurality of polymorphisms are identified which meetthese criteria for a single degenerate capture probe and a singlerestriction enzyme. For example, a plurality of polymorphisms may beidentified that are within 500 base pairs of a degenerate priming sitefor the following sequence: 5′-ACNNGTNNNNAATT-3′ (SEQ ID NO. 1) andwithin 500 base pairs of a NotI site so that the polymorphism is betweenthe NotI site and the priming site.

In one embodiment two or more collections of target sequences that varyin the common or consensus sequence may be simultaneously analyzed. Thepolymorphisms to be analyzed may each be near one of two or moredegenerate locus specific priming sites. The sites may vary in thecommon sequence, for example. Two or more separate pools of captureprobes may be synthesized and used for MARA.

In one embodiment genomic DNA is digested with a restriction enzyme andligated to an adaptor. The adaptor has a common priming site that may bedifferent from the common priming site in the degenerate capture probe.The adaptor ligated genomic DNA and the degenerate capture probes aremixed under conditions that allow hybridization of the degeneratecapture probes to corresponding priming sites in the adaptor ligatedgenomic DNA. The degenerate capture probes are then extended so that thecomplement of the adaptor sequence is incorporated into the extendeddegenerate capture probes. The extended degenerate capture probes arethen amplified by, for example, PCR using primers to the common primingsite from the adaptor and the common priming site in the degeneratecapture probes. See, FIG. 4. The amplified MARA product may then beanalyzed to determine the genotype of the one or more SNPs present. Inone embodiment an array of probes is designed to genotype SNPs predictedto be present in the MARA product.

Feature specific degenerate probes may be synthesized. The probes may besynthesized on an array using combinatorial chemistry such that eachfeature has a unique sequence but all of the features share some commonsequence. The synthesis steps for the common sequence do not require amask since all of the probes have the same base at that position. Forexample if the degenerate sequence is 5′-GTTNCNNAT-3′ all of the probeswould have the GTT-C-AT sequence and addition of these bases could bedone en masse without a photolithography step. The probes at differentfeatures would vary at the N positions but within a feature most probeswould have the same base at a given N position.

In many embodiments of the present methods one or more enrichment stepmay be included to generate a sample that is enriched for extendedcapture probes prior to amplification with common sequence primers. Insome embodiments it is desirable to separate extended capture probesfrom fragments from the starting nucleic acid sample, adaptor-ligatedfragments, adaptor sequences or non-extended capture probes, forexample. In one embodiment the capture probes are extended in thepresence of a labeled dNTP, for example dNTPs labeled with biotin. Thelabeled nucleotides are incorporated into the extended capture probesand the labeled extended capture probes are then separated fromnon-extended material by affinity chromatography. When the label isbiotin the labeled extended capture probes can be isolated based on theaffinity of biotin for avidin, streptavidin or a monoclonal anti-biotinantibody. In one embodiment the antibody may be coupled to protein-Aagarose, protein-A sepharose or any other suitable solid support knownin the art. Those of skill in the art will appreciate that biotin is onelabel that may be used but any other suitable label or a combination oflabels may also be used, such as fluorescein which may be incorporatedin the extended capture probe and an anti-fluorescein antibody may beused for affinity purification of extended capture probes. Other labelssuch as, digoxigenin, Cyanine-3, Cyanine-5, Rhodamine, and Texas Red mayalso be used. Antibodies to these labeling compounds may be used foraffinity purification. Also, other haptens conjugated to dNTPs may beused, such as, for example, dinitrophenol (DNP).

In another embodiment capture probes that have been extended through theadaptor sequence on the adaptor modified DNA are made double stranded byhybridizing and extending a primer. Only the fully extended captureprobes will have the priming site so partially extended capture probeswill remain single-stranded. The sample is then digested with a nucleasethat selectively digests single stranded nucleic acid, such as E. ColiExonuclease I. The sample is then amplified.

In another embodiment extension products may be enriched bycircularization followed by digestion with a nuclease such asExonuclease VII or Exonuclease III. The extended capture probes may becircularized, for example, by hybridizing the ends of the extendedcapture probe to an oligonucleotide splint so that the ends arejuxtaposed and ligating the ends together. The splint will hybridize tosequences in the extended capture probe and bring the 5′ end of thecapture probe next to the 3′ end of the capture probe so that the endsmay be ligated by a ligase, for example DNA Ligase or AmpligaseThermostable DNA. See, for example, U.S. Pat. No. 5,871,921 which isincorporated herein by reference. The circularized product will beresistant to nucleases that require either a free 5′ or 3′ end.

A variety of nucleases may be used in one or more of the embodiments.Nucleases that are commercially available and may be useful in thepresent methods include: Mung Bean Nuclease, E. Coli Exonuclease I,Exonuclease III, Exonuclease VII, T7 Exonuclease, BAL-31 Exonuclease,Lambda Exonuclease, RecJ_(f), and Exonuclease T. Different nucleaseshave specificities for different types of nucleic acids making themuseful for different applications. Exonuclease I catalyzes the removalof nucleotides from single-stranded DNA in the 3′ to 5′ direction.Exonuclease I degrades excess single-stranded primer oligonucleotidefrom a reaction mixture containing double-stranded extension products.Exonuclease III catalyzes the stepwise removal of mononucleotides from3′-hydroxyl termini of duplex DNA. A limited number of nucleotides areremoved during each binding event, resulting in coordinated progressivedeletions within the population of DNA molecules. The preferredsubstrates are blunt or recessed 3′-termini, although the enzyme alsoacts at nicks in duplex DNA to produce single-strand gaps. The enzyme isnot active on single-stranded DNA, and thus 3′-protruding termini areresistant to cleavage. The degree of resistance depends on the length ofthe extension, with extensions 4 bases or longer being essentiallyresistant to cleavage. This property can be exploited to produceunidirectional deletions from a linear molecule with one resistant(3′-overhang) and one susceptible (blunt or 5′-overhang) terminus.Exonuclease VII is a single-strand directed enzyme with 5′ to 3′- and 3′to 5′-exonuclease activities making it the only bi-directional E. coliexonuclease with single-strand specificity. The enzyme has no apparentrequirement for divalent cation, and is fully active in the presence ofEDTA. Initial reaction products are acid-insoluble oligonucleotideswhich are further hydrolyzed into acid-soluble form. The products oflimit digests are small oligomers (dimers to dodecamers). For additionalinformation about nucleases see catalogues from manufacturers such asNew England Biolabs, Beverly, Mass.

In some embodiments one of the primers added for PCR amplification ismodified so that it is resistant to nuclease digestion, for example, bythe inclusion of phosphorothioate. Prior to hybridization to an arrayone strand of the double stranded fragments may be digested by a 5′ to3′ exonuclease such as T7 Gene 6 Exonuclease.

In some embodiments the nucleic acid sample, which may be, for example,genomic DNA, is fragmented, using for example, a restriction enzyme,DNase I or a non-specific fragmentation method such as that disclosed inU.S. patent application Ser. No. 09/358,664, which is incorporatedherein by reference in its entirety. Adaptors containing at least onepriming site are ligated to the fragmented DNA. Locus-specific primersare synthesized which contain a different adaptor sequence at the 5′end. The adaptor-ligated genomic DNA is hybridized to the locus-specificprimers and the locus specific primer is extended. This may be done forexample, by the addition of DNA polymerase and dNTPs. Extension productsmay be amplified with primers that are specific for the adaptorsequences. This allows amplification of a collection of many differentsequences using a limited set of primers. For example, a single set ofprimers may be used for amplification. In another embodiment a secondamplification step is carried out using the same or different primers.

In some embodiments the amplified products are analyzed by hybridizationto an array of probes attached to a solid support. In some embodimentsan array of probes is specifically designed to interrogate a collectionof target sequences. The array of probes may interrogate, for example,from 1,000, 5,000, 10,000 or 100,000 to 2,000, 5,000, 10,000, 100,000,1,000,000 or 3,000,000 different target sequences. In one embodiment thetarget sequences contain SNPs and the array of probes is designed tointerrogate the allele or alleles present at one or more polymorphiclocation. The array may comprise a collection of probes that hybridizespecifically to one or more SNP containing sequences. The array maycomprise probes that correspond to different alleles of the SNP. Oneprobe or probe set may hybridize specifically to a first allele of aSNP, but not hybridize significantly to other alleles of the SNP and asecond probe set may be designed to hybridize to a second allele of aSNP but not hybridize significantly to other alleles. A hybridizationpattern from the array indicates which of the alleles are present in thesample. An array may contain probe sets to interrogate, for example,from 1,000, 5,000, 10,000 or 100,000 to 2,000, 5,000, 10,000, 100,000,1,000,000 or 3,000,000 different SNPs.

In another embodiment an array of probes that are complementary to tagsequences present in the capture probes is used to interrogate thetarget sequences. In some embodiments the amplified targets are analyzedon an array of tag sequences, for example, the Affymetrix GenFlex® array(Affymetrix, Inc., Santa Clara, Calif.). In this embodiment the captureprobes comprise a tag sequence that is unique for each species ofcapture probe. A detectable label that is indicative of the allelepresent at the polymorphic site of interest is associated with the tag.The labeled tags are hybridized to the one or more arrays and thehybridization pattern is analyzed to determine which alleles arepresent.

In another embodiment methods for generating a plurality of differentoligonucleotides are disclosed. Oligonucleotides may be synthesized inparallel on a solid support. Combinatorial chemistry may be used togenerate degeneracy in the capture probe pool. The oligonucleotides arethen released from the solid support and used for further analysis. Thereleased probes may be used, for example, for multiplex PCRamplification of a collection of target sequences, for probes, forprimers for reverse transcription or amplification or for any other useof oligonucleotides known in the art. In one embodiment theoligonucleotides on the solid support comprise a collection of captureprobes.

In another embodiment kits that are useful for the present methods aredisclosed. In one embodiment a kit for amplifying a collection of targetsequences is disclosed. The kit may comprise one or more of thefollowing: a collection of capture probes as disclosed, one or moreadaptor, one or more generic primers for common sequences, one or morerestriction enzymes, buffer, one or more polymerase, a ligase, buffer,dNTPs, ddNTPs, and one or more nucleases. The restriction enzyme of thekit may be a type-IIs enzyme. The capture probes may be attached to asolid support.

CONCLUSION

Methods are disclosed for genotyping a collection of polymorphisms usinglocus specific capture probes. The locus specific capture probes aresynthesized as degenerate oligonucleotides, by for example,combinatorial chemistry. The use of degenerate probes allows for morecost effective probe synthesis. The polymorphisms may be selected basedon proximity to a selected restriction site and proximity to adegenerate priming site. Capture probes may be synthesized on an arrayin a 3′ to 5′ direction and released from the array prior to beingextended. Alternatively the probes may be synthesized in a 5′ to 3′direction and extended on the array.

All publications and patent applications cited above are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication or patent application were specifically andindividually indicated to be so incorporated by reference. Although thepresent invention has been described in some detail by way ofillustration and example for purposes of clarity and understanding, itwill be apparent that certain changes and modifications may be practicedwithin the scope of the appended claims.

1.-41. (canceled)
 42. A method for amplifying a collection of targetsequences from a nucleic acid sample wherein each target sequencecomprises a common sequence and a consensus sequence immediatelyadjacent to the common sequence, the method comprising: fragmenting thenucleic acid sample into fragments with a selected restriction enzyme;ligating an adaptor to the fragments to obtain adaptor-ligatedfragments, wherein said adaptor comprises a first priming sequence;contacting the adaptor-ligated fragments with probes, wherein each probecomprises a second priming sequence and a target specific sequence thatcomprises a region that is complementary to the common sequence and aregion that is complementary to the consensus sequence; extending atleast some of the probes; and amplifying the extended probes withprimers to said first and second priming sequences.
 43. The method ofclaim 42, wherein labeled nucleotides are incorporated into the extendedprobes and extended probes are isolated by affinity chromatography. 44.The method of claim 43, wherein said labeled nucleotides are labeledwith biotin and, wherein avidin, streptavidin or an anti-biotin antibodyis used to isolate extended probes.
 45. The method of claim 42, whereinprior to amplification the extended probes are made double stranded andsingle stranded nucleic acid in the sample is digested with a nuclease.46. The method of claim 42, wherein there are 1,000 to 5,000 differenttarget sequences in the collection of target sequences.
 47. The methodof claim 42, wherein there are 2,000 to 10,000 different targetsequences in the collection of target sequences.
 48. The method of claim42, wherein there are 10,000 to 1,000,000 different target sequences inthe collection of target sequences.